1. Field of the Invention
This invention relates in general to the field of microscopy and, in particular, to a novel approach for providing multi-spectral illumination for pathologic analysis of tissue slides.
2. Description of the Prior Art
Changes in the cellular structure of tissue are used to detect pathologic changes, such as to assess the progress of precancerous conditions and to detect cancer. A tissue sample removed from a patient is typically sectioned and fixed to a slide for staining and microscopic examination by a pathologist. The morphology of the tissue (the visually perceptible structure and shape of features in the tissue) is analyzed to provide a qualitative assessment of its condition and to identify the presence of pathologic changes that may indicate progression towards a malignancy. For many decades, this visual procedure has been the diagnostic mainstay of pathology.
With the advent of computers and sophisticated digital imaging equipment, researchers have extended the realm of histopathology through the use of mechanized procedures for diagnostic and quantitative investigation. In such mechanized procedures, histopathologic sections and/or cytologic preparations are imaged with a microscope, and the images are digitized, stored, and analyzed for nuclear-placement patterns (histometry) or for the spatial and statistical distribution patterns of nuclear chromatin (karyometry). Karyometric assessment is always preceded by image segmentation, whereby each nucleus in an image is identified, outlined, isolated and stored as a separate image.
Each stain provides information on the localization of particular molecules in tissue or associated with different structural elements of the tissue section (e.g., nuclei). Therefore, a detailed knowledge of every stain's spatial distribution offers a distinct perspective about the biological material under review. Accordingly, localization of diverse stains in a single of tissue is a challenge of growing significance.
The conventional approach has been to employ at most two stains (e.g., hemotoxylin and eosin) in every tissue section because the human eye is often not capable of distinguishing pattern produced by additional stains. Increasing the number of stains can be problematic also for computerized analysis because of overlap between the stains' absorbance spectra. A conventional imaging system that captures a red, green, and blue (“RGB”) image of the tissue section does not provide, for example, information that may be used unambiguously to separate the absorbance contributions of each stain at a detector pixel. Therefore, the use of more than two stains in the same tissue sample would not necessarily provide additional information with conventional microscopes even through mechanized analysis.
A desirable approach would be to stain the tissue sample with additional stains and collect more than three spectral channel images (colors) at every pixel, thereby providing sufficient spectral sampling to uniquely and unambiguously separate the absorbance contributions of each stain to each pixel of the detector. However, this approach has been difficult to implement with good results because of practical tradeoffs between source brightness, detector sensitivity and resolution, optics magnification, speed of acquisition, and computational requirements.
Systems for brightfield and fluorescence multispectral recording of specimens have been described (see U.S. Pat. No. 6,690,466 and U.S. Pat. No. 6,373,568 and are commercially available. Three different approaches are used to collect the multispectral information. For example, spectral filtering is used in the Nuance™ system sold by Cambridge Research and Instrumentation, Inc. (“Cri”), of Woburn, Mass.; scanning optical-path difference (OPD) in the interferometer SpectraView® sold by Applied Spectral Imaging, Inc., of Vista, Calif.; and diffraction grating in the LSM 510 META microscope manufactured by Carl Zeiss, Inc., of Germany. CRi's system has a spectral source constructed of multiple-color LEDs that can be individually controlled, thus allowing for an illumination source of adjustable spectrum. The light source can be used to either illuminate the sample with a sequence of “pure wavelengths” using one type of LED at a time or using a mixture of LEDs simultaneously.
All existing systems have in common the use of commercial microscope objectives and remain constrained by the field-of-view/numerical-aperture trade-off associated with these optical elements. These systems are primarily intended for collecting images of a single objective field of view rather than whole-slide scanning. Accordingly, these microscope optics enable two main image-acquisition approaches, by the so-called “step-and-repeat” and “push-broom” scanning modes. In the step-and-repeat approach, individual image fields (also known as “tiles”) are recorded and the stage bearing the microscope slide is advanced to the next field, as illustrated in FIG. 1. For a typical 20× objective of approximately NA=0.75, this approach results in several thousand image fields for a 20×50 mm2 slide, the exact number depending on how much field overlap is provided and on whether or not a complete record of the slide is made. The step-and-repeat approach is necessary when spectral data are acquired by a sequence of filters or by an interferometer; otherwise, the movement of the specimen will cause inconsistency in the collected spectral measurements.
Pushbroom scanning is based on the use of a linear detector array covering the diameter of field of view of the microscope objective. The approach results in a swath that could cover the entire length of a slide, to be repeated, after a step-over, typically 20-30 times to cover a 20 mm-wide slide. This approach is illustrated in FIG. 2.
A problem with both these image-acquisition approaches is that they require an overlap between the individual tiles or swath scans to ensure complete imaging coverage of the region of interest. Thus, stitching of image tiles or swaths and additional computation are required to determine the extent of redundant image data. Therefore, a more straightforward approach to generating multi-spectral images of tissue slides would represent a useful advance in the art. The present invention utilizes an array microscope to advantageously improve multi-spectral imaging of tissue slides.